1. Field of the Invention
The invention relates to the field of liquid chromatographic analysis. More particularly the invention relates to temperature control in liquid chromatographic apparatus. By way of further characterization but not by way of limitation thereto, the invention is an apparatus for quickly reducing the temperature of the liquid flowing through the reactor of a liquid chromatography apparatus if conditions occur in the system which are conductive to boiling of the liquid.
2. Description of the Related Art
Amino acid analysis is a specialized application of a liquid column chromatographic separation technique which utilizes an ion-exchange resin as the stationary phase, with eluting buffers of varying pH and salt concentrations employing as the moving phase. Amino acids contained in a sample introduced onto the top of the chromatographic column are separated from each other as they are eluted through the resin bed which comprises the column packing. The amino acids present in the eluted stream are detected by combining the column effluent with a reagent which is metered into the stream at a fixed flow rate. The reagent, upon combining with amino acids present in the stream, forms compounds which, when subjected to a development process, can be detected by specific changes in optical properties such as absorbance or fluorescence. In the classical method developed by Stein and Moore, the reagent used is Ninhydrin dissolved in a suitable solvent/buffer solution. The development process consists of heating the solution at a fixed temperature for a specified period of time. The resulting compound has a specific color, the optical density of which is proportional to the concentration of the compound present in the flowing stream. The physical property measured is then optical absorbance measured at a specific wavelength of light.
Calibration of an amino acid analyzer in terms of its specific sensitivity to detect amino acids is directly related to the color development process. That is, optimum color development requires maintaining the eluent/reagent mixture at an elevated temperature for a fixed period of time. In order for the instrument calibration to be stable, two parameters of the development process, temperature and exposure time, must remain stable. Prior devices have accomplished this by causing the effluent to pass through a capillary coil which has been suspended in a boiling water bath. The temperature of the liquid in the coil is determined by the boiling point of the water and development time is determined by the internal volume of the capillary and the volumetric flow rate of the liquid passing therethrough. Such a device is utilized in the apparatus disclosed in U.S. Pat. No. 3,010,798 issued to E. C. Whitehead et al. on Nov. 28, 1961. While a boiling water reactor provides a stable, low cost, thermostatting device, the exposure times required necessitate the use of long capillary coils. Thus, the clear resolution between amino acids as they are eluted from the column become diffused as they are pumped through these long capillaries. In addition, a long start-up time is necessary to bring the water to a boil.
Exposure of the eluent/reagent mixture to higher temperatures for a shorter period of time, as by using shorter capillary coils, will result in maximum sensitivity and improved resolution. However, as the temperature of the reaction coil is raised, care must be taken to prevent boiling of the liquid passing through the coil. Vapor bubbles formed in the line due to boiling may pass into the cuvette of the photometer which is used for the optical detection. The presence of vapor bubbles in a cuvette destroys the calibration of the instrument and causes large noise transients significantly affecting the accuracy of the instrument. One common cause for boiling is a drop in the flow rate of buffer or reagent in the system. This drop may be caused by pump failure, by exhaustion of the supply of the substance, etc. The change in composition of the eluent/reagent mixture changes the boiling point of the substance and may lower it sufficiently to allow boiling to occur. This problem necessitates the inclusion of a means for controlling the temperature of the reagent/eluent mixture to prevent vaporization of the mixture.
A device which incorporates such a system is shown in U.S. Pat. No. 3,806,321 issued to E. L. Durrum et al. on Apr. 23, 1974. That device provides a means for quenching the heat of the mixed materials in the coil in the event that they are overheated. In that device, when it is desired to cool the coil, a cooling liquid is forced from a reservoir below the coil into a reservoir above the coil. The cooling liquid is then allowed to drain over the coil and into the reservoir below the coil. A gas under pressure is connected to the lower reservoir and is used to force the liquid into the upper reservoir. While suited for its intended purpose, this device is limited in that it cannot be cycled very often because the cooling liquid does not cool fast enough to allow for repeated uses. In addition, the liquid must be contained in the reservoirs and requires elaborate containing equipment.